Detection platforms that are adaptable to numerous target types with easy-to-read signal outputs are attractive because of their potential to be used as real-time sensors. A longstanding biosensor and the gold standard for real-time end user devices is the home glucose meter. Central to this device is the enzyme glucose oxidase (GOx), which upon recognition of its substrate results in the production of hydrogen peroxide that is easily measured via electrochemical methods. The redox properties of GOx are dependent on the presence of its co-factor flavin adenine dinucleotide (FAD). The removal of FAD reversibly renders the enzyme inactive without permanently denaturing the enzyme structure. Thus, addition of FAD can be used to 'turn-on' the activity of the enzyme. The long-term goal of this project is to develop novel electrochemical biosensors based on aptamer based target recognition linked with a modulated GOx signaling system. The marriage between these two existing strategies will produce an adaptable detection system that is easily amenable to numerous target types with the real-time signaling capabilities of the GOx signaling system. The objective of this SC2 pilot application is to generate preliminary data showing efficacy of the proposed enzyme reactivated signaling strategy in tandem with aptamer-based molecular recognition. The central hypothesis of this application, which we will pursue via two separate but complementary strategies (Aim 1 and 2 respectively), is that a target- binding event via an aptamer probe will result in the direct release of an enzyme activation trigger. The specific aims for this project are to establish, a detection strategy based on aptamer target binding events for small molecules (Aim 1) and for oligonucleotide target sequences (Aim 2). In both aims, a target-aptamer binding event will release a trigger (either modified or unmodified FAD), which will activate the GOx signaling system. In order to generate preliminary data and prove efficacy of the proposed strategies we will utilize tryptophan (an essential amino acid and important biochemical precursor) as a small-molecule target for Aim 1 and miR-21 (an important oncogenic microRNA implicated in the progression of numerous cancers) as the target sequence for Aim 2. In both aims, we will use enzyme based colorimetric assays (measurable via spectroscopic experiments) to determine the initial rates for the activated enzymes. Subsequently we will measure the binding affinity of the target (i.e. trigger) molecules to their respective aptamers via isothermal titration calorimetry. We will also carry out important control experiments to determine the analytical viability of these detection strategies with respect to selectivity and sensitivity. This proposal is innovative because it develops the first examples of using aptamer-based molecular recognition to directly turn-on the activity of attenuated GOx (apo-GOx). This work is significant because it will provide the preliminary data necessary to show proof-of-concept and will enable us to adapt the current research strategy to an electrochemical solid scaffold at the SC1 level with the long-term vision of developing real-time biosensors for multi-target detection. As importantly it will provide the appropriate mentoring needed to aid the development of the PI to the next level of being an independent researcher and faculty member.